An HIV-1 Mini Vaccine Induced Long-lived Cellular and Humoral Immune Responses

Memory formation is the most important aspect of a vaccine which can guarantee long-lasting immunity and protection. The main aim of the present study was to evaluate the memory immune responses after immunization with a mini vaccine. Mice were immunized with human immunodeficiency virus-1 P24-Nef fusion peptide and then cellular and humoral immune responses were evaluated. In order to determine long-lived memory, immune responses were monitored for 20 weeks after final immunization. The results showed that the candidate vaccine induced proliferation and cytotoxic T lymphocyte responses and shifted cytokine patterns to T helper-1 profile. Evaluation of humoral immune responses also showed an increase in total peptide specific-IgG titer and a shift to IgG2a humoral response. Monitoring of immune responses at weeks 4, 12 and 20 after last immunization showed that immunologic parameters have been sustained for 20 weeks. Our findings support the notion that long-lived memory responses were achieved using a mini vaccine immunization.

Herein, clustering of epitopes from different parts of structural and regulatory proteins of HIV particle could be of use (7,8). The construction of immunodominant epitopes from HIV-1 is a strategy that could enable a strong stimulation of humoral and cellular immune responses. As a result, now it is accepted that targeting of both humoral and cellular immune responses are needed for induction of protection against HIV infection (9). The critical role of CD8+ T cells in control of viral replication in human and non human primate models is unavoidable (10). In fact, the control of viral load initially needs the induction of strong cytotoxic T lymphocyte responses (10,11). Targeting of the cytotoxic T lymphocyte (CTL) response to certain epitopes of HIV proteins has been shown to be useful in controlling infection (12). Studies in HIV infected patients show that CTL responses directed against HIV P24 and Nef proteins, correlate with disease progression and the intensity of the immune response is directly linked to the prevention of illness progression (13). Nef is a regulatory protein and has a key role in viral replication, pathogenicity and escape from immune responses. Analyzes of HIV sequences revealed that this protein, as well as P24, has multiple conserved and immunogenic epitopes that are recognized by cytotoxic and T helper lymphocytes (14)(15)(16)(17). So, considering those properties and bioinformatic analysis results, fusion of P24 and Nef for candidate vaccine design was considered (9

Statistical analyzes
The data were expressed as Mean± SD. All statistical analyzes were done by one-way ANOVA followed by Tukey's test. In all of the cases, P value less than 0.05 was considered to be statistically significant.

Lymphoproliferative activity
Lymphocyte proliferative responses were

Cytotoxic activity
The CTL response was monitored, as a key mediator in the control of HIV infection. To detect cytotoxic activity, Granzyme B ELISPOT method was used. Analysis of GrB producing cells revealed that peptide immunization of mice increased this population compared to control groups (P=0.0001) in 50:1 of E:T ratio (Fig. 2). Monitoring of cytotoxic activity of mice immunized with P24-Nef peptide after 20 weeks revealed a decrease in CTL activity over time but significant differences were observed between immunized and control groups (P=0.0001) and no significant difference was observed between control groups (P>0.05).

IL-4 and IFN-γ assay
As shown in Fig. 3A, peptide immunization of mice significantly (P=0.0001) increased IFN-γ producing lymphocytes compared to control groups. This response has exhibited significance increase until the 20 th week of study (P<0.005). No significant difference was observed between control groups (P>0.05). We also evaluated IL-4 producing lymphocytes as Th2 cytokine marker. Evaluation of IL-4 producing cells revealed that after peptide immunization of mice, IL-4 producing lymphocytes had increased significantly (P=0.0001) compared to control groups (Fig. 3B) and this difference was stable during the study after the 20 th week of final immunization (P=0.0001). There was no significant difference between control groups (P> 0.05).

Humoral immune responses and isotyping
Humoral immune response was determined with indirect ELISA method. The results of total antibody titration showed that immunization of experimental groups with adjuvanted P24-Nef fusion peptide induced specific antibody (Fig.4a) responses. The peak of specific IgG antibody level was observed at weeks 4 (P= 0.0001) and 12 (P=